PROJECT SUMMARY Proteolytic pathways are involved in virtually every human disease including, cancer, thrombosis, arthritis, infection, inflammation, and Alzheimer's disease. Many of these cleavage events take place at the interface between cells, and they regulate intracellular interactions, especially those driven by the matrix metalloproteinases (MMPs). More than 40,000 papers have been published on these proteases, and ?MMP? is topic of more than 400 currently funded NIH grants. This massive body of work contains important information, but at this point any single publication on MMPs advances our knowledge only incrementally. In fact, for the ?popular? MMPs, much of the information being collected is conflicting, raising more questions than answers. On the other hand though, substrates have not even been identified for nearly one-half of the MMP family. The long-term objective of this project is to shift the paradigm in MMP research by making a major advance in our understanding of the structure-function relationships that guide substrate selectivity of the MMPs. To accomplish this objective we will study MMP structure and function at the systems level. Rather than trying to understand how one MMP cleaves a substrate, we will quantitatively describe how every protease in the family recognizes and cleaves its substrates. The Specific Aims of the project are to test the following hypotheses: 1) that each specificity determining position within the MMP catalytic domain makes a different contribution to substrate selectivity, 2) that physiologic (and pathophysiologic) substrates of the MMPs can be predicted by placing knowledge on subsite specificity at the catalytic cleft into physiologic context, 3) that the MMP hemopexin (HPX) domain modulates but does not alter substrate recognition and selectivity. The outcome of the study will provide the essential foundation for understanding MMP function in biology and pathology at the systems level. The study will reveal the structural basis for specialization and expansion of function, which remains one of the most important unanswered questions in biology. The work will also reveal a host of new therapeutic targets because the substrates are the real downstream effectors of MMP activity. Additionally, the study will generate peptide substrates that are highly selective for individual matrix metalloproteinases. These substrates can be used to devise agents to image proteolysis in animals and in humans. The same substrates could also be employed to create targeted drug delivery vehicles. Finally, all information derived from the study, computational tools, results, predictions, proteomic analysis and interpretations will be deposited on our Proteolysis MAP website (www.proteolysis.org) for use by the scientific and medical community.